False positive, promiscuous ligands dominate hit lists and are a key problem in drug discovery screens. Here we consider a single mechanism that might unite our understanding of most promiscuous hits: compound aggregation followed by receptor sequestration. Many molecules, including hits, leads, reagents, and even some drugs, can aggregate, and when they do so their properties change dramatically, often perniciously but sometimes beneficially. We investigate the prevalence of aggregators in biologically relevant libraries, determine how they act, and develop rapid screens to detect them. The specific aims are: 1. To explore the prevalence of aggregators in screening and their effect on biological assays. We begin with simple-but-essential questions: what percentage of screening libraries form promiscuous aggregates? What percentage of all false-positives owe to aggregation? What range of receptors will they inhibit? We will screen large, drug-like screening libraries composed of hundreds-of-thousands of molecules for aggregators, using an assay developed in the last period. We will characterize every class of false positive, not only aggregates, in detail. We will explore whether aggregation affects membrane-bound receptors, in addition to enzymes. A more complex series of questions investigates effects on downstream biological assays. Aggregates disrupt membranes and are hemolytic, and it is appropriate to wonder how they might affect toxicological assays. We will also investigate whether aggregates can persist among orally-dosed drugs, playing a role in their distribution, as has been suggested by others. 2. To investigate the mechanism of aggregation-based inhibition. The exact mechanism of aggregation-based inhibition remains unclear. For instance, we do not understand why aggregate association leads to enzyme inhibition. Does the aggregate denature the enzyme, or is the effect more subtle, perhaps affecting dynamic motion? What is the structure of aggregates;do they resemble vesicles or solid colloids? What is their affinity for protein? A combination of simple techniques, such as centrifugation, gel electrophoresis, and enzymology, and higher resolution techniques, such as electron microscopy and deuterium-exchange mass spectroscopy, will be used to investigate these questions. PUBLIC HEALTH RELEVANCE: High throughput screening is the most widely used technique to discover new drug leads, but unfortunately, these screens are dominated by false hits. Most of these false hits are explained by the tendency of many organic molecules to aggregate in solution, and so disrupt assays. By understanding and controlling aggregation, this project attempts to address this problem, and so improve drug discovery